1. Field of Invention
The invention relates to methods for treating cocaine abuse. More particularly, the invention relates to anti-cocaine vaccines that elicit immune responses for reducing the psychoactive effects of cocaine consumption.
2. Background
Cocaine is a powerfully addictive substance and new strategies are needed to treat its abuse. Cocaine degrades spontaneously in vitro and in vivo (E. R. Garrett, et al., Journal of Pharmaceutical Science (1983): vol. 72, p 258-271; D. J. Stewart, et al., Clin. Pharmacol. Ther. (1979): vol. 25, p 464-468). A principle route for the degradation of cocaine is the hydrolysis of the methyl ester to produce the nonpsychoactive compound benzoylecgonine (K. A. Cunningham, et al., Neuropsychopharmacology (1990): vol 3, p 41-50). Nonspecific esterases are also known to contribute to the in vivo degradation of cocaine through cleavage of both the methyl and benzoate esters (M. R. Brzezinski, et al., Biochem. Pharmacol. (1994): vol. 48, p 1747-1755; C. S. Boyer, et al., J. Pharmacol. Exp. Ther. (1992): vol. 260, p 939-946; R. A. Dean, et al. FASEB J. (1991): vol. 5, p 2735-2739; K. Matsubara, et al., Forensic Sci. Intl.(1984): vol. 26, p 169-180; Y. Liu, et al., J. Chromatography (1982): vol. 248, p 318-320).
Donald Landry, et al. disclose that catalytic monoclonal antibodies (mAbs) directed to the hydrolysis of cocaine can be elicited by immunization with transition state analogues of cocaine (PCT International Application, WO 9320076 A1, published Oct. 14, 1993 based upon serial number 93-PCT/US 3163, filed Apr. 2, 1993; and Science (1993): vol. 259, p 1899-1901). The catalytic mAbs generated thereby are shown by Landry to catalyze the hydrolysis of cocaine and to reduce cocaine levels in human blood thereby. The catalytic mAbs are further disclosed by Landry to be therapeutically employable for treating cocaine overdose and/or cocaine addiction.
G. P. Basmadjian, et al., disclose that catalytic polyclonal antibodies directed to the hydrolysis of cocaine can be elicited by immunization with transition state analogues of cocaine (Chem. Pharm. Bull. (1995): vol. 43, p 1902-1911). Preliminary results showed that mice immunized with immunoconjugates derived from these analogues produced, in some cases, high titers of serum catalytic antibodies as judged from an in vitro radioassay. No further work has been reported.
S. Spector, et al. disclose that mice can be actively immunized with a morphine immunogen and that serum from such mice contain antibodies that bind dihydromorphine. Morphine effects and the plasma concentration of morphine were shown to be diminished in these immunized mice (S. Spector, et al., Pharmacol. Rev. (1973): vol. 25, p 281-291; and B. Berkowitz, et al., Science (1972): vol. 178, p 1290-1292).
What is needed is an anti-cocaine vaccine for generating an active immunization to cocaine. More particularly, the antibodies generated by the anti-cocaine vaccine should block the actions of the cocaine by preventing the entry of cocaine into the central nervous system. The anti-cocaine vaccine should be characterized by reduced side effects as compared to the side effects associated with treatments based on manipulation of central neurotransmitter function.
It is disclosed herein that generating an active immunization to cocaine offers a means of blocking the actions of the drug by preventing it from entering the central nervous system. This method of treatment has less side effects than treatments based on manipulation of central neurotransmitter function. The design and preparation of a cocaine immunogen requires special regard for the stability of cocaine both free and as a haptenic determinant. Immunochemistry and a well-defined behavioral paradigm are brought together to address the problem of inactivation of the psychostimulant actions of cocaine. Active immunization is achieved with a novel, stable cocaine conjugate, disclosed below, which suppresses locomotor activity and stereotyped behavior in subjects induced by cocaine but not by amphetamine. Moreover, following acute injection of cocaine, levels of cocaine in the striatum and cerebellum of the immunized subjects are significantly lower than those of control animals. These results demonstrate that immunopharmacotherapy can be employed for treating cocaine abuse.
The design and preparation of a cocaine immunogen requires special attention to the stability of free cocaine in solution and as a haptenic determinant. Cocaine degrades spontaneously in vitro and in vivo. (E. R. Garrett, et al., J. Pharmaceutical Sci. (1983): vol. 72, p 258-271; and D. J. Stewart, et al., Clin. Pharmacol. Ther. (1979): vol. 25, p 464-468. Degradation occurs largely through hydrolysis of the methyl ester to produce the nonpsychoactive compound benzoylecgonine (K. A. Cunningham, et. al., Neuropsychopharmacol.(1990): vol. 3, p 41-50). Nonspecific esterases are also known to contribute to the in vivo degradation of cocaine through cleavage of both the methyl and benzoate esters (M. R. Brzezinski, et. al., Biochem. Pharmacol. (1994): vol. 48, p 1747-1755; C. S. Boyer, et. al., J. Pharmacol. Exp. Ther. (1992): vol. 260, p 939-946; R. A. Dean, et. al. FASEB J. (1991): vol. 5, p 2735-2739; K. Matsubara, et al., Forensic Sci. Intl.(1984): vol. 26, p 169-180; Y. Liu, et. al., J. Chromatography (1982): vol. 248, p 318-320). Conjugates that display epitopes structurally similar to those of metabolites, especially benzoylecgonine, would compromise the avidity and specificity of a cocaine-specific immune response (M. J. Taussig, Current Topics Microbiol. Immunol. (1973): vol. 60, p 125-174; and A. L. Misra, et al., Res. Comm. Che. Path. Pharmacol. (1976): vol. 13, p 579-584). Also, an appreciable benzoylecgonine titer would be exceptionally detrimental since the antiserum would be inadequate in neutralizing cocaine, particularly in the presence of rapidly formed and stable metabolites. Although each retains the phenyl ring as a major recognition element, the neutrality of cocaine contrasts with the negatively charged benzoylecgonine, a factor in antibody binding, making it possible to maximize the affinity and selectivity for cocaine. By joining the carrier protein to the cocaine framework using a linker at the position occupied by the methyl ester, any minor decomposition of the linked hapten results not in a benzoylecgonine response, but primarily in nonhaptenic recognition. Attention to such aspects of the immunochemistry must be emphasized in view of an unsuccessful report of a potential cocaine prophylactic (O. Bagasra, et al., Immunopharmacol.(1992): vol. 23, p 173-179; G. Gallacher, Immunopharmacol (1994): vol. 27, p 79-81). The hapten 4 (compound 4) was synthesized in four steps starting from (xe2x88x92)-cocaine (FIG. 1). The key reaction, alkylation of (xe2x88x92)-ecgonine, introduced the required tether. The stereochemical configuration remained intact at C-2 of the tropane nucleus. This ester linker mimics the alkyl character of the methyl ester of cocaine which is important for recognition of this part of the molecule. Coupling of 4 to keyhole limpet hemocyanin (KLH) afforded the conjugate, 4-KLH, for immunization.
One aspect of the invention is directed to cocaine analogs. Preferred cocaine analogs are represented by the following structures: 
where n is greater than or equal to 2 and less than or equal to 8. In preferred embodiments, n is greater than or equal to 4, less than or equal to 6, or equal to five.
Another aspect of the invention is directed to cocaine immunoconjugates. Preferred cocaine immunoconjugates are represented by the following structure: 
where n is greater than or equal to 2 and less than or equal to 8. In preferred embodiments, n is greater than or equal to 4, less than or equal to 6, or equal to five. An additional preferred cocaine immunoconjugate is represented by the following structure: 
wherein n and m are greater than or equal to 4, less than or equal to 6, or n is six and m is four.
Another aspect of the invention is directed to a method for suppressing psychoactive effects of cocaine within a subject. The method includes a step wherein an anti-cocaine vaccine is administered to the subject. The anti-cocaine vaccine is of a type which includes an injectable sterile solvent and an immunogenic amount of a cocaine immunoconjugate. Preferred immunoconjugates are indicated above.
Another aspect of the invention is directed to an anti-cocaine vaccine. The anti-cocaine vaccine comprises a sterile injectable medium and a cocaine immunoconjugate admixed with said sterile injectable medium. Preferred immunoconjugates are indicated above.
Another aspect of the invention is directed to a method for reducing psychoactive effects displayed by a subject upon administration of cocaine. The method comprises two steps. In the first step, an anti-cocaine immune response is elicited within the subject by vaccination with an anti-cocaine vaccine. The anti-cocaine vaccine includes one of the cocaine immunoconjugates indicated above. In the second step, cocaine is administered to the subject.
Another aspect of the invention is directed to a method for obtaining anti-cocaine polyclonal antibodies. The method includes two steps, an anti-cocaine immune response is elecited within a subject by vaccination with an anti-cocaine vaccine. The anti-cocaine vaccine includes one of the cocaine immunoconjugates indicated above. In the second step, anti-cocaine polyclonal antibodies are isolated from the subject.
Another aspect of the invention is directed to anti-cocaine polyclonal antibodies produced according to the method indicated above.
An other apsect of the invention is directed to a method for obtaining anti-cocaine monoclonal antibodies. The method includes three steps. In the first step, an anti-cocaine immune response is elicited within a subject by vaccination with an anti-cocaine vaccine. The anti-cocaine vaccine includes one of the cocaine immunoconjugates indicated above. In the second step, an antibody producing cell from the subject of said Step A which expresses an anti-cocaine antibody is isolated and cloned. In the third step, anti-cocaine monoclonal antibody expressed by antibody producing cell isolated and cloned in the second step are isolated.
Another aspect of the invention is directed to anti-cocaine monoclonal antibodies produced according to the method indicated above.